Kinetics and mechanism of the oxidation of the glutathione dimer by hypochlorous acid and catalytic reduction of the chloroamine product by glutathione reductase

Oxidized glutathione (GSSG) reacts with two molar equivalents of HOCl/OCl- (a neutrophil-derived oxidant and a common biocide) to form the dichloro (bis-N-chloro-gamma-L-glutamyl) derivative (NDG). The reaction of less than two molar equivalents of HOCl with GSSG does not yield the unsymmetrical monochloro derivative (NCG) but rather a stoichiometric amount of NDG and GSSG. This result is explained by a faster reaction of the second equivalent of HOCl with NCG than that of the first equivalent of HOCl with GSSG. The rates of reaction of GSSG(2-), GSSG(3-), and GSSG(4-) (successive deprotonation of the ammonium groups) have been investigated, and it is clear that GSSG(2-) is unreactive, whereas GSSG(4-) is about twice as reactive as GSSG(3-). Accordingly, the following mechanism is proposed (constants for 5 degrees C): H+ + OCl- = HOCl, pK(1) = -7.47; GSSG(2-) = GSSG(3-) + H+, pK(2) = 8.5; GSSG(3-) = GSSG(4-) + H+, pK(3) = 9.5; GSSG(3-) + HOCl -> NCG(3-) + H2O, k(4) = 2.7(2) x 10(6) M-1 s(-1); GSSG(4-) + HOCl -> NCG(4-) + H2O, k(5) = 3.5(3) x 10(7) M-1 s(-1); NCG(3-) -> NDG(4-) + H+, k(6) = fast; and NCG(4-) + HOCl -> NDG(4-) + H2O, k(7) = fast. At physiologic pH, the k(4) pathway dominates. NDG decomposes at pH 7.4 in a first-order process with k(dec) = 4.22(1) x 10(-4) s(-1) (t(1/2) = 27 min). Glutathione reductase (EC 1.6.4.2) is capable of catalyzing the reduction of NDG by NADPH. The only NDG-derived product that is observed (by NMR) after the reduction by NADPH is GSH. Thus, in the presence of the GOR/NADPH system, GSH is capable of redox buffering a 3/2 mol equiv of HOCl rather than a 1/2 mol equiv as previously assumed.